Normal embryonic development involves the concerted action of gene products regulated in a stage-specific manner. A model system widely used in the study of expression of such genes is the embryonal carcinoma (EC) cell system. F9 EC cells resemble embryonal ectoderm and the differentiated derivatives resemble embryonic parietal endoderm. We have isolated a gene whose mRNA synthesis is regulated during EC cell differentiation and early embryogenesis. This gene encodes an endogenous intracisternal A particle (IAP) and can act as a transposon, inerting itself to activate oncogenes, in turn, leading to cellular transformation. Eukaryotic genes xgcontain cis-regulatory elements that, through interaction with multiple classes of trans-acting cellular proteins, mediate enhanced, repressed or basal transcription rates. Thus, characterization of cis regulatory elements of the IAP gene and of the cellular proteins that regulate expression of the gene during EC cell differentiation should provide information on the genetic control of embryogenesis and c-oncogene activation. Toward this objective, we have grossly defined the putative enhancer or repressor and promoter regions of the IAP gene and have shown that cellular proteins from expressing as well as non- expressing cells bind to the putative enhancer. In this application, we propose to identify and characterize cis-elements and transacting factors participating in positive or negative regulation of IAP transcription. We will identify enhancer and promoter regions of the IAP gene, their cis-regulatory elements, the regulatory proteins interacting with the cis-elements and then determine whether the proteins are transcription activators or repressors. The activator or repressor will be isolated and used to raise antisera for screening cDNA clones encoding the IAP transcription regulatory proteins. The cloned cDNA will be sequenced to determine its nucleotide sequence and open reading frames. Based on these data, subfragments of cDNA encoding subregions of the regulatory proteins will be recloned into expression vectors for production of partial peptides to be used in studies of functional domains. The results should facilitate our understanding of how transcription regulators mediate stage- specific gene expression during embryogenesis.